The thyroid gland is part of a critical endocrine circuit that influences development and oxidative metabolism in humans, which is largely conserved amongst the vertebrates. As the basic hormone-forming unit of the gland, each thyroid follicle is comprised of a monolayer of epithelial cells secreting thyroglobulin (Tg) into a central apical cavity (the follicle lumen). Thyrocytes access iodide using an electrochemical gradient at the basolateral membrane to drive intracellular accumulation of iodide with eventual transport apically where, in conjunction with H2O2 generation and peroxidase activity, Tg becomes iodinated to form both iodotyrosines and iodothyronines. Ultimately, endocytosis and proteolytic digestion allows for release of active thyroid hormones to the bloodstream while deiodination of iodotyrosines reclaims iodide for future Tg iodination. This new grant cycle focuses on Tg transport through the secretory pathway. In Tg, simple structural requirements for hormonogenesis are counterbalanced by evolutionary pressures to conserve iodide, which has resulted in a complex secretory glycoprotein. This complexity generates several fundamental questions concerning intracellular protein transport and endoplasmic reticulum (ER) quality control function that link directly to clinical disease. In the overview, our objective in this grant remains to pursue a basic science analysis in order to explain the most clinically relevant phenotypes. Our primary focus in this renewal application centers around the Tg protein and how difficulties in its intracellular transport relate to patients and animal models with thyroid disease including congenital hypothyroidism with and without goiter. Our Specific Aims for the next 5 years are to elucidate the role of the cholinesterase-like (ChEL) domain of Tg in thyroid hormone biosynthesis, and to develop an in vivo paradigm in which to study cell biological defects associated with congenital Tg deficiency. These studies have clinical significance specific to thyroidology while providing more general insight into the basic and clinical problems found in endoplasmic reticulum storage diseases. PUBLIC HEALTH RELEVANCE: The thyroid gland is part of a critical endocrine circuit that influences development and metabolism in humans. To make thyroid hormone, thyroid follicle cells secrete thyroglobulin (Tg) into a central cavity that functions as a storage depot for iodide and for thyroid hormones, because both iodotyrosines and and thyroid hormones are formed and stored within the Tg protein. Iodinated Tg thus serves the dual function of both thyroid hormone reservoir and iodide reservoir for the body. Tg transport through the secretory pathway is required for its iodination and storage of thyroid hormones. One group reports that mutations in the gene encoding Tg are the most common cause of congenital hypothyroidism (other than iodide deficiency which threatens 200 million peple worldwide). Of the Tg mutants characterized to date, all are defective for transport through the secretory pathway, being retained in a compartment known as the endoplasmic reticulum (ER). Our primary focus centers on the Tg protein and its relationship to patients and animal models with thyroid disease including, but not limited to, congenital goiter.